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Analysis of the Structural Elements Required for Highly Efficient Catalysis

高效催化所需的结构元素分析

基本信息

批准号：
01470149
负责人：
OHNO Motonori
金额：
$ 3.26万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

A highly reactive Asp-49-phospholipase A_2 (PLA_2) and two Lys-49-PLA_2, whose activities are only 1-2% that of Asp-49-PLA_2, from Trimeresurus flavoviridis venom contain 122 amino acid residues, and their sequences are 60% homologous and the positions of the 14 half-cystine residues are identical. Such PLA_2 isozymes provide an invaluable system for elucidating the structural elements required for eliciting catalytic function and various physiological functions. The aim of this research is to study the structure-function relationships of PLA_2 through synthesis of mutant proteins of the types of T. flavoviridis PLA_2. (1) As a step of this study, an active site His47-Asp99 couple in Asp-49-PLA_2 was investigated by pH-dependent methylation of His47 with methyl p-nitrobenzenesulfonate. Protonation of His47 appears to stabilize a transient state of catalysis. (2) Three cDNAs for Asp-49-PLA_2 and two Lys-49-PLA_2 were prepared. The amino acid sequence deduced from the uncleotide sequence … More of cDNA of Asp-49-PLA_2 was partially inconsistent with the amino acid sequence determined with the protein. The reinvestigation of the protein sequence established that the sequence from cDNA is correct. The nucleotide sequence of a cDNA encoding a PLA_2 which is similar to an Asp-49-PLA_2 called PL-X was also sequenced. Expression of the proteins using the cDNAs by recombinant DNA technology has not yet been successful. Intensive study is in progress. (3) Comparison of the four cDNAs encoding four different PLA_2 led to an interesting discovery that the nucleotide sequences of 5'- and 3'-noncoding regions are much more homologous than those of the coding regions. As a step for searching the roles of the noncoding regions in regulation and expression, analysis of genome DNA is on the way. One of nine clones obtained us under analysis. (4) Although expression of PLA_2 proteins has not yet been successful during the period, the finding of the unique nucleotide sequences for T. flavoviridis PLA_2 isozymes established the basis for future development. Less

竹叶青蛇毒中一种活性很高的Asp-49-磷脂酶A_2（PLA_2）和两种活性仅为Asp-49-PLA_2的1-2%的Lys-49-PLA_2均含有122个氨基酸残基，它们的序列同源性为60%，其中14个半胱氨酸残基的位置完全相同。这种PLA_2同工酶为阐明催化功能和各种生理功能所需的结构元件提供了一个宝贵的系统。本研究的目的是通过合成T. flavoviridis PLA_2. (1)作为本研究的一个步骤，通过对硝基苯磺酸甲酯对His 47的pH依赖性甲基化来研究Asp-49-PLA_2中的活性位点His 47-Asp 99偶联。His 47的质子化似乎稳定了催化的瞬态。(2)制备了三个Asp-49-PLA_2和两个Lys-49-PLA_2的cDNA。从核苷酸序列推导的氨基酸序列 ...更多信息 Asp-49-PLA_2的cDNA序列与蛋白质测定的氨基酸序列部分不一致。对蛋白质序列的重新研究确定了cDNA序列是正确的。另外还测定了编码PLA_2的cDNA的核苷酸序列，该PLA_2与一个称为PL-X的Asp-49-PLA_2相似。使用cDNA通过重组DNA技术表达蛋白质尚未成功。密集的研究正在进行中。(3)比较4种PLA_2基因的cDNA序列，发现PLA_2基因5 '和3'非编码区的核苷酸序列比编码区的同源性高得多。作为探索非编码区在调控和表达中的作用的一步，基因组DNA的分析正在进行中。九个克隆体中的一个得到了我们的信息。(4)虽然PLA_2蛋白的表达在此期间尚未成功，但T.黄绿菌PLA_2同工酶为今后的研究奠定了基础。少